Phitosterols suspension concentrates and process of preparation thereof

ABSTRACT

Concentrated suspensions containing phytosterols are described, in an amount higher than 25% by weight, in addition to a process for their preparation and their relative use as stimulants of plant growth.

The present invention relates to concentrated suspensions ofphytosterols and the preparation process thereof.

The present invention also relates to the use of said concentratedsuspensions as plant growth stimulants.

STATE OF THE ART

Sterols are a class of chemical compounds deriving from sterol, apolycyclic compound composed of four condensed rings, and they play afundamental role in the physiology of animals and plants.

When sterols are produced from plants, they are referred to asphytosterols. Among these the most important are campesterol,sitosterol, stigmasterol and the brassicasterol.

Phytosterols have a wide spectrum of use; they are present in fact incosmetics, in food products as additive for reducing cholesterol levels,but they are also known in agriculture for their marked fertilizingactivity, as described for example in patent applications CH206735,CN101948350, CN104292041 or CN104355837.

Numerous formulations containing phytosterols are known in the art foruse in both the pharmaceutical field and also in the agrochemical field.These formulations can be in the form of solid or liquid preparations,or in the form of suspensions of solids in liquids.

For a correct use, the formulations must be capable of solubilizing, ordispersing, or they must give rise to stable emulsions when diluted inappropriate volumes of water, in turn applied to crops or on the soil inwhich the crop is grown, before or after sowing or germination.

Many agropharmaceuticals are validly used in the form of concentratedsuspensions (CS), a specific type of liquid formulation in which thefinely ground active ingredient is suspended in water with the additionof particular surfactants such as wetting agents, dispersants, rheologymodifiers, antifoam agents and biocides, which improve their stabilityand make them more suitable for agricultural use.

There are numerous advantages of concentrated suspensions: a reducedenvironmental impact due to the absence of organic solvents, in additionto greater safety for the operator in applying the product to theground.

Phytosterols, however, hardly lend themselves to being formulated asconcentrated suspensions as, due to their chemical structure, they tendto increase the viscosity of the formulation and show signs of physicalinstability such as flocculation or aggregation.

Patent application CN103262849 describes aqueous suspensions comprisinggeneric phytosterols in a wide range of concentrations, withoutproviding specific examples, however, of compositions with highconcentrations of phytosterols.

Patents U.S. Pat. Nos. 3,881,005, 4,195,084 and EP1197153 indicate infact that the maximum concentration of phytosterols in an aqueoussuspension for human use is equal to about 20-25% by weight, a limitconcentration at which the suspension is described as being notexcessively viscous and easy to pour.

More recently, patent U.S. Pat. No. 8,414,945 describes a compositionwith a high content of phytosterols, in the form of powder dispersiblein water.

A similar formulation, however, has drawbacks for the agriculturaloperator due to difficulties in calculating the exact dosage to beapplied and the risk of inhalation during the preparation operations ofthe mixture.

The need for liquid formulations is therefore strongly felt in theagrochemical sector, which, having a higher concentration, allow areduced volume of the formulation to be used for reaching the dosage ofuse in a single treatment, with a consequent reduction in theproduction, transportation and disposal costs.

DESCRIPTION

Contrary to what is described above, the Applicant has now surprisinglyfound a process for the preparation of concentrated suspensionscontaining phytosterols in an amount higher than 25% by weight withrespect to the total weight of the suspension.

The suspensions obtained by this process are stable over time, with nothickening phenomena and are easy to pour and suitable for use in theagricultural sector.

A first object of the present invention therefore relates to a processfor the preparation of concentrated suspensions containing phytosterolsin an amount higher than 25% by weight with respect to the total weightof the suspension, comprising a step for grinding said phytosterols andpossible wetting agents and dispersing agents until an average particlesize of less than 10 μm is reached.

Said grinding step can be carried out on the phytosterols, alone ormixed with wetting agents and dispersing agents or other co-formulants,or on the final concentrated suspension containing phytosterols, wettingagents and dispersing agents and other co-formulants dissolved ordispersed in water.

Said grinding allows a particle size of the particles to be reached,preferably equal to or less than 10 μm for Dv 50 and 30 μm for Dv 90.

The term “Dv 50” represents the value of the average diameter belowwhich there is 50% of the particles.

The term “Dv 90” represents the value of the average diameter belowwhich there is 90% of the particles.

As is well known to skilled persons in the field, said grinding iseffected at a controlled temperature ranging from 15 to 45° C.

The preparation of the above-mentioned concentrated suspensions iseffected according to known methods, by mixing the components in thedesired ratios and in any order.

The preparation process according to the present invention allows aconcentrated suspension of phytosterols to be obtained in an amounthigher than 25% by weight of phytosterols with respect to the totalweight of the suspension, surprisingly with respect to what is describedin the state of the art.

This process can also be used for concentrated suspensions containingphytosterols in an amount lower than 25% by weight with respect to thetotal weight of the suspension, also in this case providing a stable andpourable formulation and therefore suitable for use in the agriculturalfield.

A further object of the present invention therefore relates to aconcentrated suspension comprising:

a) at least one phytosterol in an amount higher than 25% by weight withrespect to the total weight of the concentrated suspension;b) one or more wetting agents in an amount ranging from 1% to 5% byweight with respect to the total weight of the concentrated suspension;c) one or more dispersing agents in an amount ranging from 1% to 5% byweight with respect to the total weight of the concentrated suspension.

Said phytosterols are selected from alpha-sitosterol, beta-sitosterol,stigmasterol, ergosterol, campesterol, alpha-spinosterol, brassicasteroland mixtures thereof.

Said phytosterols are preferably selected from beta-sitosterol,stigmasterol, campesterol, brassicasterol and mixtures thereof. Thephytosterols in the concentrated suspension according to the presentinvention are more preferably a mixture of beta-sitosterol,stigmasterol, campesterol and brassicasterol.

In the above mixture of phytosterols, the beta-sitosterol is present inan amount ranging from 40% to 45% by weight, the campesterol is presentin an amount ranging from 20% to 25% by weight, the stigmasterol ispresent in an amount ranging from 20% to 30% by weight and thebrassicasterol is present in an amount ranging from 1% to 5% by weight,with respect to the total weight of the mixture of phytosterols.

In the above-mentioned mixture of phytosterols, the beta-sitosterol ispresent in an amount of about 43% by weight, the campesterol is presentin an amount of about 24% by weight, the stigmasterol is present in anamount of about 26% by weight and the brassicasterol is present in anamount of about 2% by weight, with respect to the total weight of themixture of phytosterols.

Said mixture of phytosterols is present in the concentrated suspensionof the invention in an amount higher than 25% by weight, preferably inan amount ranging from 26% to 40% by weight, more preferably rangingfrom 26% to 35% by weight, with respect to the total weight of theconcentrated suspension.

Said one or more wetting agents are selected from non-ionic surfactants,anionic surfactants and mixtures thereof, said one or more wettingagents are preferably anionic surfactants.

The non-ionic surfactants used as wetting agents are preferably selectedfrom esters of sorbitan and ethoxylated fatty alcohols.

The anionic surfactants used as wetting agents are preferably selectedfrom sulfosuccinates and sulfonated alkylnaphthalene and their salts;they are more preferably sulfosuccinates and salts thereof, and evenmore preferably a sulfosuccinate sodium salt.

According to the present invention, said one or more wetting agents arepresent in the concentrated suspension in an amount ranging from 1% to5% by weight, preferably from 2% to 4% by weight, and are morepreferably equal to about 3% by weight, with respect to the total weightof the concentrated suspension.

Said one or more dispersing agents are selected from non-ionicsurfactants, anionic surfactants and mixtures thereof, said one or moredispersing agents are preferably anionic surfactants.

The non-ionic surfactants used as dispersing agents are preferablyselected from ethoxy-propoxylated block copolymers, acrylic polymers,polycarboxylates, methyl oleyl taurates, tristyrylphenol ethoxylates,tristyrylphenol ethoxy-propoxylates, tristyrylphenol phosphateethoxylates, tristyrylphenol sulfate ethoxylates, alkyl polyglucosidesand mixtures thereof.

The anionic surfactants used as dispersing agents are preferablyselected from tristyrylphenol phosphate ethoxylates, tristyrylphenolsulfate ethoxylates, condensed alkylnaphthalene sulfonates,ligninosulfonates and mixtures thereof. They are more preferablyselected from condensed alkylnaphthalene sulfonates andligninosulfonates.

According to the present invention, said one or more dispersing agentsare present in the concentrated suspension in an amount ranging from 1%to 5% by weight, preferably from 1.5% to 3% by weight, more preferablyequal to about 2% by weight, with respect to the total weight of theconcentrated suspension.

As is well known to skilled persons in the field, the concentratedsuspensions of the inventions can also comprise further surfactants andco-formulants, such as rheology modifiers, antifoam agents andbiocides/preservatives.

Optionally, further compounds can also be added to the aboveconcentrated suspensions for improving the biological activity.

Examples of these compounds are mineral salts, such as for example,compounds of nitrogen, phosphorous, potassium and known mixtures thereofsuch as NPK, algae, humic acids, fulvic acids, stillage, molasses.

The complement to 100 consists of the solvent, i.e. water.

The concentrated suspensions according to the present invention alsoshow a high biostimulating activity, intended as a stimulant of plantgrowth and do not show any phytotoxicity with respect to the applicationcrops. These characteristics make them suitable for use in theagricultural field in the stimulation of plant growth.

A further object of the present invention therefore relates to the useof the above-mentioned concentrated suspensions as stimulants of plantgrowth.

The Applicant has in fact verified that these concentrated suspensionsare capable of stimulating the metabolism of plants also in the presenceof stress (cold, salinity of the soil), determining the development andenlargement of the fruit and also preventing senescence of the plant.

The concentrated suspensions according to the present invention can beapplied to the crop via the leaves, or to the soil by means offertigation, or incorporation into the ground, or through seed tanning.

Examples of crops of interest are fruit trees, vegetables and extensivecrops such as rice and wheat.

A further object of the present invention therefore relates to a methodfor stimulating plant growth in cultivated areas, which consists inapplying effective and non-phytotoxic doses of said concentratedsuspensions, on any part of the plant.

The amount of formulation to be applied in order to obtain the desiredeffect can vary according to various factors such as, for example, thephytosterols used, the crop, the climatic conditions, thecharacteristics of the soil, the application method, etc. Overalldosages of formulations ranging from 50 ml to 4 l per hectare ofagricultural crop generally provide a sufficient activity.

The following examples are now provided for a better understanding ofthe invention, which should be considered as being illustrative andnon-limiting of the same.

Example 1

Components % (by weight) Mixture of phytosterols 31.6 Antifoam agent 1Biocide 0.09 Imbirol OT/Na 70 3 Supragil MNS 88 2 Water 62.31 TOTAL 100

The mixture of phytosterols refers to the mixture of beta-sitosterol,campesterol, stigmasterol and brassicasterol, previously indicated asthe preferred mixture: the beta-sitosterol is present in an amount equalto about 43% by weight, the campesterol is present in an amount equal toabout 24% by weight, the stigmasterol is present in an amount equal toabout 26% by weight and the brassicasterol is present in an amount equalto about 2% by weight, with respect to the total weight of the mixtureof phytosterols.

The antifoam agent is a polydimethylsiloxane.

The biocide is2,2′,2″-(hexahydro-1,3,5-triazin-1-1,3,5-triyl)triethanol.

Imbirol OT/Na 70 corresponds to the sodium salt of adialkylsulfosuccinate.

Supragil MNS 88 corresponds to the sodium salt ofmethylnaphthalenesulfonic acid.

Example 2

Component % (by weight) Mixture of phytosterols 31.6 Antifoam agent 1Biocide 0.09 Imbirol OT/Na 70 3 Supragil MNS 90 2 Water 62.31 TOTAL 100

The mixture of phytosterols refers to the mixture of beta-sitosterol,campesterol, stigmasterol and brassicasterol, previously indicated asthe preferred mixture: the beta-sitosterol is present in an amount equalto about 43% by weight, the campesterol is present in an amount equal toabout 24% by weight, the stigmasterol is present in an amount equal toabout 26% by weight and the brassicasterol is present in an amount equalto about 2% by weight, with respect to the total weight of the mixtureof phytosterols.

The antifoam agent is a polydimethylsiloxane.

The biocide is 2,2′,2″-(hexahydro-1,3,5-triazin-1,3,5-triyl)triethanol.

Imbirol OT/Na 70 corresponds to the sodium salt of adialkylsulfosuccinate.

Supragil MNS 90 corresponds to the sodium salt ofmethylnaphthalenesulfonic acid, polymer with formaldehyde.

Example 3

Component % (by weight) Mixture of phytosterols 31.6 Antifoam agent 1Biocide 0.09 Imbirol OT/Na 70 3 Vanisperse CB 2 Water 62.31 TOTAL 100

The mixture of phytosterols refers to the mixture of beta-sitosterol,campesterol, stigmasterol and brassicasterol, previously indicated asthe preferred mixture: the beta-sitosterol is present in an amount equalto about 43% by weight, the campesterol is present in an amount equal toabout 24% by weight, the stigmasterol is present in an amount equal toabout 26% by weight and the brassicasterol is present in an amount equalto about 2% by weight, with respect to the total weight of the mixtureof phytosterols.

The antifoam agent is a polydimethylsiloxane.

The biocide is2,2′,2″-(hexahydro-1,3,5-triazin-1-1,3,5-triyl)triethanol.

Imbirol OT/Na 70 corresponds to the sodium salt of adialkylsulfosuccinate Vanisperse CB corresponds to a sodiumligninosulfonate.

The formulations of Examples 1-3 were subjected to the acceleratedstability test (according to Cipac MT 46.1) which aims at acceleratingthe aging of the product by heating. This test serves to mimic thestability of a specific formulation after 2 years of storage at roomtemperature.

The procedure is the following: a certain quantity of formulation (forexample 500 mL) is inserted in a hermetically closed container which isleft in an oven at 54° C. for 14 days. After this period of time, thecontainer is removed from the oven and is left to return to roomtemperature. It is then verified that the formulation has not undergonephase separation or other phenomena of physical instability such asflocculation, aggregation or crystalline growth.

Example 4

Pea Test Application to the Soil Production Evaluation

The effect on the final production of dwarf pea plants of twoformulations CS at 31.6%, one containing the formulation of Example 1and the other the same formulation not ground, was evaluated afterapplication to the soil.

Seeds of “Meraviglia d'Italia” dwarf peas were sown in pots having adiameter of 15 cm, suitably filled with a mixture of earth and soilpreviously sterilized.

The first treatment was carried out 12 days after sowing, pouring aquantity of 400 g/ha of formulation at the collar of the plant. Each potcontained 5 seedlings and there were 4 replicas for each thesis. Theplants were kept in a greenhouse, at a temperature of 24° C. during theday and 18° C. overnight, photoperiod: 16 hours of light and 8 hours ofdarkness. A total of 4 treatments were carried out during theexperiment, at 10-day intervals.

The final survey provided for the evaluation of the number of pods thathad not yet reached maturity, the number of ripe pods, the number ofseeds contained in them and their dry weight. The flowers still presenton the plant at the time of harvesting were also counted.

Average Average Average Average Nr. DRY Nr. Nr. of Average of WT. OF ofRIPE UNRIPE Nr. PRODUCTS FLOWERS SEEDS* SEEDS SEEDS of SEEDS UTC 1.251.04 3.50 4.25 5.00 CS 31.6% 1.00 1.78 6.00 0.50 9.50 NOT groundFormulat. 1.00 3.05 6.50 0.75 14.00 Exa. 1

The results obtained showed the positive effect of the two formulationswhich induced the maturation of a greater number of pods with aconsequent increase in the number of seeds produced. Between the twoformulations tested, that containing the ground technical productaccording to the invention gave the best results.

Example 5 Cucumber Test Application to the Soil Evaluation of the PlantVigour

The effect on the biomass of cucumber plants of two formulations CS at31.6%, one containing the formulation of Example 1 and the other thesame formulation not ground, was evaluated after application to thesoil.

“Long China” cucumber seedlings were transplanted at the stage of 2 realleaves, in pots having a diameter of 11 cm, suitably filled with amixture of earth and soil previously sterilized. The treatment wascarried out the day after the transplantation, pouring a quantity offormulation equal to a dose of 400 g/ha at the collar of the plant. Eachpot contained 1 seedling and 5 replicas were present for each thesis.The plants were kept in an air-conditioned cell, at a temperature of 24°C. photoperiod: 16 hours of light and 8 hours of darkness.

The final survey provided for the evaluation of the fresh weight of thegreen biomass, carried out two weeks after the transplantation.

PRODUCTS Average WEIGHT (g) WEIGHT sum (g) UTC 13.55 67.74 Formulationof 14.59 72.95 Example 1 CS 31.6% 12.53 62.64 NOT ground

The results obtained showed the positive effect of the formulationcontaining the ground technical product according to the presentinvention: the plants treated with this product, in fact, had a moredeveloped biomass with respect to the blank and the formulationcontaining the non-ground technical product.

Example 6 Cucumber Test Leaf Application Evaluation of the Plant Vigour

The effect on the biomass of cucumber plants of two formulations CS at31.6%, one containing the formulation of Example 1 and the other thesame formulation not ground, was evaluated after application to theleaves.

“Long China” cucumber seedlings were transplanted at the stage of 2 realleaves, in pots having a diameter of 11 cm, suitably filled with amixture of earth and soil previously sterilized. The treatment wascarried out the day after the transplantation, spraying the leaves witha quantity of 6 g/hl, using a sprayer machine. Each pot contained 1seedling and 5 replicas were present for each thesis. The plants werekept in an air-conditioned cell, at a temperature of 24° C.,photoperiod: 16 hours of light and 8 hours of darkness.

2 treatments were carried out during the experiment, at 5-day intervals.

The final survey provided for the evaluation of the fresh weight of thegreen biomass, carried out two weeks after the transplantation.

Average WEIGHT PRODUCTS WEIGHT (g) sum (g) UTC 15.18 75.9 CS 31.6%16.214 81.07 NOT ground Formulation Example 1 16.838 84.19

The results obtained showed the positive effect of the formulationcontaining the ground technical product according to the presentinvention, also when applied via the leaves: the plants treated withthis product, in fact, had a more developed biomass with respect to theblank and the formulation containing the non-ground technical product.

1. A process for the preparation of concentrated suspensions containingphytosterols in an amount higher than 25% by weight with respect to thetotal weight of the suspension, said process comprising a grinding stepof said phytosterols and possible wetting agents and dispersing agentsuntil an average particle size of less than 10 μm is reached.
 2. Theprocess according to claim 1, wherein the grinding step is carried outon phytosterols, alone or in a mixture with wetting agents anddispersing agents, or on the final concentrated suspension containingphytosterols, wetting agents, dispersing agents and other co-formulantsdispersed in water.
 3. The process according to claim 1, whereinphytosterols and possible wetting agents and dispersing agents have aparticle size equal to 10 μm for Dv 50 and 30 μm for Dv
 90. 4. Theprocess according to claim 1, wherein the grinding step is carried outat a temperature ranging from 15° C. to 45° C.
 5. A concentratedsuspension comprising: a) a phytosterol mixture of beta-sitosterol,stigmasterol, campesterol and brassicasterol, in an amount higher than25% by weight with respect to the total weight of the concentratedsuspension; b) one or more wetting agents in an amount ranging from 1%to 5% by weight with respect to the total weight of the concentratedsuspension; c) one or more dispersing agents in an amount ranging from1% to 5% by weight with respect to the total weight of the concentratedsuspension.
 6. The suspension according to claim 5, wherein thephytosterols are a mixture of beta-sitosterol, stigmasterol, campesteroland brassicasterol wherein the beta-sitosterol is present in an amountranging from 40% to 45% by weight, the campesterol is present in anamount ranging from 20% to 25% by weight, the stigmasterol is present inan amount ranging from 20% to 30% by weight and the brassicasterol ispresent in an amount ranging from 1% to 5% by weight, with respect tothe total weight of the mixture of phytosterols.
 7. The suspensionaccording to claim 6, wherein the beta-sitosterol is present in anamount equal to about 43% by weight, the campesterol is present in anamount equal to about 24% by weight, the stigmasterol is present in anamount equal to about 26% by weight and the brassicasterol is present inan amount equal to about 2% by weight, with respect to the total weightof the mixture of phytosterols.
 8. The suspension according to claim 5,wherein one or more wetting agents are selected from non-ionicsurfactants, anionic surfactants and mixtures thereof.
 9. The suspensionaccording to claim 5, wherein one or more dispersing agents are selectedfrom non-ionic surfactants, anionic surfactants and mixtures thereof.10. Use of a concentrated suspension according to claim 5, as plantgrowth stimulants.
 11. A method for stimulating plant growth incultivated areas, which consists of applying effective andnon-phytotoxic doses of a concentrated suspension according to claim 5,on any part of the plant.
 12. The suspension according to claim 5wherein the phytosterol mixture of beta-sitosterol, stigmasterol,campesterol and brassicasterol is in an amount ranging from 26% to 40%by weight of the concentrated suspension.
 13. The suspension accordingto claim 5 wherein the phytosterol mixture of beta-sitosterol,stigmasterol, campesterol and brassicasterol is in an amount rangingfrom 26% to 35% by weight with respect to the total weight of theconcentrated suspension.
 14. The suspension according to claim 5 whereinthe one or more wetting agents is in an amount ranging from 2% to 4% byweight with respect to the total weight of the concentrated suspension.15. The suspension according to claim 5 wherein the one or more wettingagents is in an amount equal to about 3% by weight with respect to thetotal weight of the concentrated suspension.
 16. The suspensionaccording to claim 5 wherein the one or more dispersing agents is in anamount ranging from 1.5% to 3% by weight with respect to the totalweight of the concentrated suspension.
 17. The suspension according toclaim 5 wherein the one or more dispersing agents is equal to about 2%by weight with respect to the total weight of the concentratedsuspension.
 18. The suspension according to claim 8 wherein the one ormore wetting agents are anionic surfactants.
 19. The suspensionaccording to claim 9 wherein the one or more dispersing agents areanionic surfactants.
 20. The use of a suspension according to claim 10comprising a method taken from the group consisting of leaf applicationto the crop, application to the soil by fertigation, incorporation intothe soil, seed tanning, or combinations thereof.